PROJECT SUMMARY/ABSTRACT The goal of this research is to investigate the molecular mechanisms responsible for chromosome conformation. Chromosome structure is important for controlling genomic processes such as transcription, replication, and chromosome segregation, and disruptions to this structure are found in genetic diseases and cancer cells. There are three main levels of chromosome organization: chromosome territories, chromosome compartments, and topologically associating domains (TADs). The current model is that TAD formation occurs due to dynamic loop extrusion of chromatin fibers, which is blocked in a directional manner by CTCF bound to TAD boundaries. However, the components and molecular mechanism of this proposed topological machine which forms these chromatin loops are currently unknown. Previous studies suggest that topoisomerases and histone variants may have a role in regulating chromosome conformation and topological machine activity. In addition, recent molecular modeling research has implicated loop extrusion e.g. dynamically extruded DNA loops, as an important characteristic of chromosome structure, however this has not yet been tested experimentally. This study will use genomic methods such as Hi-C, ChIP-seq, and TMP-seq in combination with cell biological and functional genetic approaches to study the molecular components and dynamics of the topological machine. Three complementary aims will be performed to address this question: 1) Assess the role of topoisomerases that have recently been identified as a part of the CTCF complex at TAD boundaries. 2) Investigate the role of histone variants that decorate key elements involved in TAD biology. 3) Develop new methods to determine chromatin dynamics inside TADs to test recently proposed models of TAD formation by dynamic loop formation. Together, completion of these aims will lead to new insights about the function and regulation of chromosome conformation.